1. Field of the Invention
The present invention relates to the identification of new muteins of fibroblast growth factor 21 that have reduced proteolytic degradation when expressed in yeast.
2. Description of the Related Art
Fibroblast growth factors are large polypeptides widely expressed in developing and adult tissues (Baird et al., Cancer Cells, 3:239-243, 1991) and play crucial roles in multiple physiological functions including angiogenesis, mitogenesis, pattern formation, cellular differentiation, metabolic regulation and repair of tissue injury (McKeehan et al., Prog. Nucleic Acid Res. Mol. Biol. 59:135-176, 1998). According to the published literature, the FGF family now consists of at least twenty-three members, FGF-1 to FGF-23 (Reuss et al., Cell Tissue Res. 313:139-157 (2003).
Fibroblast growth factor-21 (FGF-21) has been reported to be preferentially expressed in the liver (Nishimura et al., Biochimica et Biophysica Acta, 1492:203-206, (2000); WO01/36640; and WO01/18172) and described as a treatment for ischemic vascular disease, wound healing, and diseases associated with loss of pulmonary, bronchia or alveolar cell function and numerous other disorders. More recently, FGF-21 has been shown to stimulate glucose-uptake in mouse 3T3-L1 adipocytes in the presence and absence of insulin, and to decrease fed and fasting blood glucose, triglycerides, and glucagon levels in ob/ob and db/db mice and 8 week old ZDF rats in a dose-dependant manner, thus, providing the basis for the use of FGF-21 as a therapy for treating diabetes and obesity (WO03/011213).
The development of recombinant DNA technology has made possible the production of foreign products such as muteins of FGF-21 in host cells in which exogenous DNA sequences coding for those products have been introduced. The advantage of this technology is that products can be produced in high yields, in highly purified form, with low risk of contamination such as viral contamination. These recombinant techniques have been widely used for the production of recombinant proteins in prokaryotic as well as eukaryotic host cells.
However, the large-scale production of recombinant products by these techniques is still limited, due to problems of expression efficiency of these exogenous DNA sequences and also to vector instability. Recombinant products produced in heterologous eukaryotic hosts usually differ from their naturally-occurring counterpart in their glycosylation content. Moreover, recombinant products are also susceptible to intracellular proteolytic degradation by the host cell in which they are made. It is known that certain amino acid sequences are prone to degradation by proteolytic enzymes which usually has an adverse effect on protein activity. This susceptibility is due in part to the secondary and tertiary structure of the protein, exposing certain amino acids present on the surface of proteins to attack by proteolytic enzymes (Leszczynski et al., Science 234:849, 1986).
The present invention solves the problem of proteolytic degradation of FGF-21 by providing muteins of FGF-21 with reduced susceptibility for proteolytic degradation, wherein one or more protease labile amino acids are substituted by protease non-labile amino acid(s). Applicants have found that the FGF-21 muteins with reduced susceptibility for proteolytic degradation can be produced in industrial yeast fermentation conditions and maintain the biological activity necessary to be useful to treat subjects with disorders including, but not limited to, type 2 diabetes, obesity, and metabolic syndrome.